1. Field of the Invention
The invention relates to inhibitors of the nucleic enzyme poly(adenosine 5xe2x80x2-diphospho-ribose) polymerase [xe2x80x9cpoly(ADP-ribose) polymerasexe2x80x9d or xe2x80x9cPARPxe2x80x9d, which is also sometimes called xe2x80x9cPARSxe2x80x9d for poly(ADP-ribose) synthetase]. More particularly, the invention relates to the use of PARP inhibitors to prevent and/or treat neural tissue damage resulting from ischemia and reperfusion injury, neurological disorders and neurodegenerative diseases; to prevent or treat vascular stroke; to treat or prevent cardiovascular disorders; or to treat other disorders such as arthritis; diabetes; septic shock (such as endotoxic shock); inflammatory bowel disorders (such as colitis and Crohn""s disease); and cancer.
2. Description of the Prior Art
Poly(ADP-ribose) polymerase (xe2x80x9cPARPxe2x80x9d) is an enzyme located in the nuclei of cells of various organs, including muscle, heart and brain cells. PARP plays a physiological role in the repair of strand breaks in DNA. Once activated by damaged DNA fragments, PARP catalyzes the attachment of up to 100 ADP-ribose units to a variety of nuclear proteins, including histones and PARP itself. While the exact range of functions of PARP has not been fully established, this enzyme is thought to play a role in enhancing DNA repair.
During major cellular stresses, however, the extensive activation of PARP can rapidly lead to cell death through depletion of energy stores. Four molecules of ATP are consumed for every molecule of NAD (the source of ADP-ribose) regenerated. Thus, NAD, the substrate of PARP, is depleted by massive PARP activation and, in the efforts to re-synthesize NAD, ATP may also be depleted.
It has been reported that PARP activation plays a key role in both NMDA- and NO-induced neurotoxicity, as shown by the use of PARP inhibitors to prevent such toxicity in cortical cultures in proportion to their potencies as inhibitors of this enzyme (Zhang et al., xe2x80x9cNitric Oxide Activation of Poly(ADP-Ribose) Synthetase in Neurotoxicityxe2x80x9d, Science, 263:687-89 (1994); and in hippocampal slices (Wallis et al., xe2x80x9cNeuroprotection Against Nitric Oxide Injury with Inhibitors of ADP-Ribosylationxe2x80x9d, NeuroReport, 5:3, 245-48 (1993). The potential role of PARP inhibitors in treating neurodegenerative diseases and head trauma has thus been known. Research, however, continues to pinpoint the exact mechanisms of their salutary effect in cerebral ischemia, (Endres et al., xe2x80x9cIschemic Brain Injury is Mediated by the Activation of Poly(ADP-Ribose)Polymerasexe2x80x9d, J. Cereb. Blood Flow Metabol., 17:1143-51 (1997)) and in traumatic brain injury (Wallis et al., xe2x80x9cTraumatic Neuroprotection with Inhibitors of Nitric Oxide and ADP-Ribosylation, Brain Res., 710:169-77 (1996)).
It has been demonstrated that single injections of PARP inhibitors have reduced the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle in rabbits. In these studies, a single injection of the PARP inhibitor, 3-amino-benzamide (10 mg/kg), either one minute before occlusion or one minute before reperfusion, caused similar reductions in infarct size in the heart (32-42%). Another PARP inhibitor, 1,5-dihydroxyisoquinoline (1 mg/kg), reduced infarct size by a comparable degree (38-48%). Thiemermann et al., xe2x80x9cInhibition of the Activity of Poly(ADP Ribose) Synthetase Reduces Ischemia-Reperfusion Injury in the Heart and Skeletal Musclexe2x80x9d, Proc. Natl. Acad. Sci. USA, 94:679-83 (1997). This finding has suggested that PARP inhibitors might be able to salvage previously ischemic heart or skeletal muscle tissue.
PARP activation has also been shown to provide an index of damage following neurotoxic insults by glutamate (via NMDA receptor stimulation), reactive oxygen intermediates, amyloid xcex2-protein, n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite N-methyl-4-phenylpyridine (MPP+), which participate in pathological conditions such as stroke, Alzheimer""s disease and Parkinson""s disease. Zhang et al., xe2x80x9cPoly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damagexe2x80x9d, J. Neurochem., 65:3, 1411-14 (1995). Other studies have continued to explore the role of PARP activation in cerebellar granule cells in vitro and in MPTP neurotoxicity. Cosi et al., xe2x80x9cPoly(ADP-Ribose) Polymerase (PARP) Revisited. A New Role for an Old Enzyme: PARP Involvement in Neurodegeneration and PARP Inhibitors as Possible Neuroprotective Agentsxe2x80x9d, Ann. N. Y. Acad. Sci., 825:366-79 (1997) ; and Cosi et al., xe2x80x9cPoly(ADP-Ribose) Polymerase Inhibitors Protect Against MPTP-induced Depletions of Striatal Dopamine and Cortical Noradrenaline in C57B1/6 Micexe2x80x9d, Brain Res., 729:264-69 (1996).
Neural damage following stroke and other neurodegenerative processes is thought to result from a massive release of the excitatory neurotransmitter glutamate, which acts upon the N-methyl-D-aspartate (NMDA) receptors and other subtype receptors. Evidence includes findings in many animal species, as well as in cerebral cortical cultures treated with glutamate or NMDA, that glutamate receptor antagonists block neural damage following vascular stroke. Dawson et al., xe2x80x9cProtection of the Brain from Ischemiaxe2x80x9d, Cerebrovascular Disease, 319-25 (H. Hunt Batjer ed., 1997).
The stimulation of NMDA receptors, in turn, activates the enzyme neuronal nitric oxide synthase (NNOS), which causes the formation of nitric oxide (NO), which more directly mediates neurotoxicity. Protection against NMDA neurotoxicity has occurred following treatment with NOS inhibitors. See Dawson et al., xe2x80x9cNitric Oxide Mediates Glutamate Neurotoxicity in Primary Cortical Culturesxe2x80x9d, Proc. Natl. Acad. Sci. USA, 88:6368-71 (1991); and Dawson et al., xe2x80x9cMechanisms of Nitric Oxide-mediated Neurotoxicity in Primary Brain Culturesxe2x80x9d, J. Neurosci., 13:6, 2651-61 (1993). Protection against NMDA neurotoxicity can also occur in cortical cultures from mice with targeted disruption of NNOS. See Dawson et al., xe2x80x9cResistance to Neurotoxicity in Cortical Cultures from Neuronal Nitric Oxide Synthase-Deficient Micexe2x80x9d, J. Neurosci., 16:8, 2479-87 (1996).
It is known that neural damage following vascular stroke is markedly diminished in animals treated with NOS inhibitors or in mice with NNOS gene disruption. Iadecola, xe2x80x9cBright and Dark Sides of Nitric Oxide in Ischemic Brain Injuryxe2x80x9d, Trends Neurosci., 20:3, 132-39 (1997); and Huang et al., xe2x80x9cEffects of Cerebral Ischemia in Mice Deficient in Neuronal Nitric Oxide Synthasexe2x80x9d, Science, 265:1883-85 (1994). See also, Beckman et al., xe2x80x9cPathological Implications of Nitric Oxide, Superoxide and Peroxynitrite Formationxe2x80x9d, Biochem. Soc. Trans., 21:330-34 (1993). Either NO or peroxynitrite can cause DNA damage, which activates PARP. Further support for this is provided in Szabxc3x3 et al., xe2x80x9cDNA Strand Breakage, Activation of Poly(ADP-Ribose) Synthetase, and Cellular Energy Depletion are Involved in the Cytotoxicity in Macrophages and Smooth Muscle Cells Exposed to Peroxynitritexe2x80x9d, Proc. Natl. Acad. Sci. USA, 93:1753-58 (1996).
Zhang et al., U.S. Pat. No. 5,587,384, issued Dec. 24, 1996, discusses the use of certain PARP inhibitors, such as benzamide and 1,5-dihydroxy-isoquinoline, to prevent NMDA-mediated neurotoxicity and, thus, treat stroke, Alzheimer""s disease, Parkinson""s disease and Huntington""s disease. However, it is has now been discovered that Zhang et al. may have been in error in classifying neurotoxicity as NMDA-mediated neurotoxicity. Rather, it may have been more appropriate to classify the in vivo neurotoxicity present as glutamate neurotoxicity. See Zhang et al. xe2x80x9cNitric Oxide Activation of Poly(ADP-Ribose) Synthetase in Neurotoxicityxe2x80x9d, Science, 263:687-89 (1994). See also, Cosi et al., Poly(ADP-Ribose)Polymerase Inhibitors Protect Against MPTP-induced Depletions of Striatal Dopamine and Cortical Noradrenaline in C57B1/6 Micexe2x80x9d, Brain Res., 729:264-69 (1996).
It is also known that PARP inhibitors effect DNA repair generally. Cristovao et al., xe2x80x9cEffect of a Poly(ADP-Ribose)Polymerase Inhibitor on DNA Breakage and Cytotoxicity Induced by Hydrogen Peroxide and xcex3-Radiation,xe2x80x9d Terato., Carcino., and Muta., 16:219-27 (1996), discusses the effect of hydrogen peroxide and xcex3-radiation on DNA strand breaks in the presence of and in the absence of 3-aminobenzamide, a potent inhibitor of PARP. Cristovao et al. observed a PARP-dependent recovery of DNA strand breaks in leukocytes treated with hydrogen peroxide.
Evidence also exists that PARP inhibitors are useful for treating inflammatory bowel disorders. Salzman et al., xe2x80x9cRole of Peroxynitrite and Poly(ADP-Ribose)Synthase Activation Experimental Colitis,xe2x80x9d Japanese J. Pharm., 75, Supp. I:15 (1997), discusses the ability of PARP inhibitors to prevent or treat colitis. Colitis was induced in rats by intraluminal administration of the hapten trinitrobenzene sulfonic acid in 50% ethanol. Treated rats received 3-aminobenzamide, a specific inhibitor of PARP activity. Inhibition of PARP activity reduced the inflammatory response and restored the morphology and the energetic status of the distal colon. See also, Southan et al., xe2x80x9cSpontaneous Rearrangement of Aminoalkylithioureas into Mercaptoalkylguanidines, a Novel Class of Nitric Oxide Synthase Inhibitors with Selectivity Towards the Inducible Isoformxe2x80x9d, Br. J. Pharm., 117:619-32 (1996); and Szabxc3x3 et al., xe2x80x9cMercaptoethylguanidine and Guanidine Inhibitors of Nitric Oxide Synthase React with Peroxynitrite and Protect Against Peroxynitrite-induced Oxidative Damagexe2x80x9d, J. Biol. Chem., 272:9030-36 (1997).
Evidence also exists that PARP inhibitors are useful for treating arthritis. Szabxc3x3 et al., xe2x80x9cProtective Effects of an Inhibitor of Poly(ADP-Ribose)Synthetase in Collagen-Induced Arthritis,xe2x80x9d Japanese J. Pharm., 75, Supp. I:102 (1997), discusses the ability of PARP inhibitors to prevent or treat collagen-induced arthritis. See also Szabxc3x3 et al., xe2x80x9cDNA Strand Breakage, Activation of Poly(ADP-Ribose)Synthetase, and Cellular Energy Depletion are Involved in the Cytotoxicity in Macrophages and Smooth Muscle Cells Exposed to Peroxynitrite,xe2x80x9d Proc. Natl. Acad. Sci. USA, 93:1753-58 (March 1996); Bauer et al., xe2x80x9cModification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a ras-transformed Bovine Endothelial Cell Line by Treatment with 5-Iodo-6-amino-1,2-benzopyrone (INH2BP)xe2x80x9d, Intl. J. Oncol., 8:239-52 (1996); and Hughes et al., xe2x80x9cInduction of T Helper Cell Hyporesponsiveness in an Experimental Model of Autoimmunity by Using Nonmitogenic Anti-CD3 Monoclonal Antibodyxe2x80x9d, J. Immuno., 153:3319-25 (1994).
Further, PARP inhibitors appear to be useful for treating diabetes. Heller et al., xe2x80x9cInactivation of the Poly(ADP-Ribose)Polymerase Gene Affects Oxygen Radical and Nitric Oxide Toxicity in Islet Cells,xe2x80x9d J. Biol. Chem., 270:19, 11176-80 (May 1995), discusses the tendency of PARP to deplete cellular NAD+ and induce the death of insulin-producing islet cells. Heller et al. used cells from mice with inactivated PARP genes and found that these mutant cells did not show NAD+ depletion after exposure to DNA-damaging radicals. The mutant cells were also found to be more resistant to the toxicity of NO.
Further still, PARP inhibitors have been shown to be useful for treating endotoxic shock or septic shock. Zingarelli et al., xe2x80x9cProtective Effects of Nicotinamide Against Nitric Oxide-Mediated Delayed Vascular Failure in Endotoxic Shock: Potential Involvement of PolyADP Ribosyl Synthetase,xe2x80x9d Shock, 5:258-64 (1996), suggests that inhibition of the DNA repair cycle triggered by poly(ADP ribose) synthetase has protective effects against vascular failure in endotoxic shock. Zingarelli et al. found that nicotinamide protects against delayed, NO-mediated vascular failure in endotoxic shock. Zingarelli et al. also found that the actions of nicotinamide may be related to inhibition of the NO-mediated activation of the energy-consuming DNA repair cycle, triggered by poly(ADP ribose) synthetase. See also, Cuzzocrea, xe2x80x9cRole of Peroxynitrite and Activation of Poly(ADP-Ribose) Synthetase in the Vascular Failure Induced by Zymosan-activated Plasma,xe2x80x9d Brit. J. Pharm., 122:493-503 (1997).
Yet another known use for PARP inhibitors is treating cancer. Suto et al., xe2x80x9cDihydroisoquinolinones: The Design and Synthesis of a New Series of Potent Inhibitors of Poly(ADP-Ribose) Polymerasexe2x80x9d, Anticancer Drug Des., 7:107-17 (1991), discloses processes for synthesizing a number of different PARP inhibitors. In addition, Suto et al., U.S. Pat. No. 5,177,075, discusses several isoquinolines used for enhancing the lethal effects of ionizing radiation or chemotherapeutic agents on tumor cells. Weltin et al., xe2x80x9cEffect of 6(5H)-Phenanthridinone, an Inhibitor of Poly(ADP-ribose) Polymerase, on Cultured Tumor Cellsxe2x80x9d, Oncol. Res., 6:9, 399-403 (1994), discusses the inhibition of PARP activity, reduced proliferation of tumor cells, and a marked synergistic effect when tumor cells are co-treated with an alkylating drug.
Large numbers of known PARP inhibitors have been described in Banasik et al., xe2x80x9cSpecific Inhibitors of Poly(ADP-Ribose) Synthetase and Mono(ADP-Ribosyl)-Transferasexe2x80x9d, J. Biol. Chem., 267:3, 1569-75 (1992), and in Banasik et al., xe2x80x9cInhibitors and Activators of ADP-Ribosylation Reactionsxe2x80x9d, Molec. Cell. Biochem., 738:185-97 (1994).
However, the approach of using these PARP inhibitors to reduce NMDA-receptor stimulation, or to treat or prevent neural tissue damage caused by NO, ischemia and reperfusion of the heart or skeletal muscle, arthritis, diabetes, endotoxic or septic shock, inflammatory diseases of the bowel (such as colitis and Crohn""s disease), and cancer, has been limited in effect. For example, side effects have been observed with some of the best-known PARP inhibitors, as discussed in Milam et al., xe2x80x9cInhibitors of Poly(Adenosine Diphosphate-Ribose) Synthesis: Effect on Other Metabolic Processesxe2x80x9d, Science, 223:589-91 (1984). Specifically, the PARP inhibitors 3-aminobenzamide and benzamide not only inhibited the action of PARP but also were shown to affect cell viability, glucose metabolism, and DNA synthesis. Thus, it was concluded that the usefulness of these PARP inhibitors may be severely restricted by the difficulty of finding a dose small enough to inhibit the enzyme without producing additional metabolic effects.
Accordingly, there remains a need for compounds that inhibit PARP activity and compositions and methods containing PARP inhibitors are more potent and reliable with fewer side effects, particularly with respect to vascular stroke.
Further, other multicyclic, nitrogen-containing, alkoxy-substituted compounds are known:
1,3-Dihydro-4-methoxy-thieno[3,4-c]quinoline, which has the following structure: 
is disclosed in White et al., xe2x80x9cQuinoline Analogues of Ortho-Quinodimethane,xe2x80x9d Tetrahedron Letters, 36:33, 5983-86 (1995). This structure is also disclosed in White et al., xe2x80x9cDihydrothiophenes as Precursors to Fused Quinolines, Quinolones and Coumarins via o-Quinodimethane Intermediates,xe2x80x9d Tetrahedron Letters, 52:9, 3117-34 (1996). Both of the White references also disclose the following structures: 
It is not believed that the above disclosed compounds have been shown to inhibit PARP activity per se.
The compounds of the invention have formula I: 
or a pharmaceutically acceptable salt, prodrug, metabolite, optical isomer or stereoisomer thereof, wherein:
R1, when present, is hydrogen or lower alkyl;
R2 is lower alkyl, aryl, aralkyl, lower alkanoyl, or xe2x80x94(CH2)nxe2x80x94(CHOH)y(CH2)mA, wherein n is 1-4, y is 0 or 1, m is 0-5, and A is cycloalkyl, cycloalkenyl, lower alkanoyl, aryl, aralkyl, xe2x80x94NH2, xe2x80x94NH-(lower alkyl), 
Y represents the atoms necessary to form a fused 5- to 6-membered ring that is aromatic or nonaromatic and carbocyclic or heterocyclic;
Z is
(i) xe2x80x94CHR2CHR3xe2x80x94 where R2 and R3 are independently hydrogen, alkyl, aryl or aralkyl;
(ii) xe2x80x94R6Cxe2x95x90CR3xe2x80x94 where R6 and R3 are independently hydrogen, lower alkyl, aryl, aralkyl, chlorine, bromine or xe2x80x94NR7R8, where R7 and R8 are independently hydrogen or lower alkyl, or, R6 and R3, taken together, form a fused 5- to 6-membered ring that is aromatic or nonaromatic and carbocyclic or heterocyclic;
(iii) xe2x80x94R2Cxe2x95x90Nxe2x80x94;
(iv) xe2x80x94CR2(OH)xe2x80x94NR7xe2x80x94; or
(v) xe2x80x94C(O)xe2x80x94NR7xe2x80x94;
provided that:
when R6 and R3 form a fused benzene ring, then Y is neither (a) a fused, 6-membered, nonaromatic carbocyclic ring nor (b) a fused, 5-membered, nonaromatic heterocyclic ring having a sulfur atom as its sole heteroatom.
In another embodiment, a process of making the compound of formula I: 
or a pharmaceutically acceptable salt, prodrug, metabolite, optical isomer or stereoisomer thereof, wherein R1, R2, n, y, m, A, Y, Z, R6, R3, R7, and R8 are as defined above, comprises the step of contacting an intermediate having formula II: 
with R1X wherein X is a bromo, chloro or iodo moiety.
In yet another embodiment, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable carrier and a compound of formula I: 
or a pharmaceutically acceptable salt, prodrug, metabolite, optical isomer or stereoisomer thereof, wherein:
R1, when present, is hydrogen or lower alkyl;
R2 is lower alkyl, aryl, aralkyl, lower alkanoyl, or xe2x80x94(CH2)nxe2x80x94(CHOH)y(CH2)mA, wherein n is 1-4, y is 0 or 1, m is 0-5, and A is cycloalkyl, cycloalkenyl, lower alkanoyl, aryl, aralkyl, xe2x80x94NH2, xe2x80x94NH-(lower alkyl), 
Y represents the atoms necessary to form a fused 5- to 6-membered ring that is aromatic or nonaromatic and carbocyclic or heterocyclic;
Z is
(i) xe2x80x94CHR2CHR3xe2x80x94 where R2 and R3 are independently hydrogen, alkyl, aryl or aralkyl;
(ii) xe2x80x94R6Cxe2x95x90CR3xe2x80x94 where R6 and R3 are independently hydrogen, lower alkyl, aryl, aralkyl, chlorine, bromine or xe2x80x94NR7R8, where R7 and R8 are independently hydrogen or lower alkyl, or, R6 and R3, taken together, form a fused 5- to 6-membered ring that is aromatic or nonaromatic and carbocyclic or heterocyclic;
(iii) xe2x80x94R2Cxe2x95x90Nxe2x80x94;
(iv) xe2x80x94CR2(OH)xe2x80x94NR7xe2x80x94; or
(v) xe2x80x94C(O)xe2x80x94NR7xe2x80x94;
provided that:
when R6 and R3 form a fused benzene ring, then Y is neither (a) a fused, 6-membered, nonaromatic carbocyclic ring nor (b) a fused, 5-membered, nonaromatic heterocyclic ring having a sulfur atom as its sole heteroatom.
In particularly preferred embodiments of the above composition, the amount of the compound of formula I is present in an amount effective for inhibiting PARP activity, for effecting a neuronal activity not mediated by NMDA toxicity, or for treating arthritis, diabetes, an inflammatory bowel disorder, a cardiovascular disorder, septic shock, or cancer.
In an additional embodiments, a method of inhibiting PARP activity comprises administering a compound of formula I, as described above for the pharmaceutical compositions of the invention. In preferred embodiments, the amount of the compound administered in the methods of the invention is sufficient for effecting neuronal activity not mediated by NMDA toxicity or for treating arthritis, diabetes, inflammatory bowel disorders, cardiovascular disorders, septic shock, or cancer.